Phenolic Coatings and Methods of Making and Using Same

ABSTRACT

A method of making a facile, surface-independent, polyphenol coating is disclosed. In general, the method includes contacting at least a portion of the substrate to be coated with an aqueous solution containing one or more salts and one or more nitrogen-free phenolic compounds. Substrates of all kinds may be used, and compounds used to make the coating may include epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC) and epicatechin-3-gallate (ECG), tannic acid, gallic acid, pyrogallol, and/or other nitrogen-free phenolic compounds. The coating made using the method, methods of using the coating, and kits comprising the coating precursors are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/162,253 filed Jan. 23, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/756,029 filed Jan. 24, 2013, bothof which are incorporated by reference in their entirety for allpurposes.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Number R37DE 014193 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

The claimed invention was made as a result of activities undertakenwithin the scope of a joint research agreement between Baxter HealthcareCorporation and Northwestern University.

FIELD OF THE INVENTION

This invention is directed to polymeric coatings that are spontaneouslydeposited on a substrate surface when the substrate surface is contactedwith an aqueous solution comprising an effective amount of one or morenitrogen-free phenolic compounds.

BACKGROUND OF THE INVENTION

Modification of material surfaces plays a central role in modernchemical, biological and material sciences, as well as in appliedsciences, engineering and technology. Methods for the modification ofbulk material substrates have been developed by interfacial chemistryusing organothiol-metals, enediol-oxides, silane-oxides, and otherphysicochemical methods, in which the predominant purpose is to imposedesired properties on non-functional substrates. Molecules utilized forsurface modification mostly have bifunctional end groups in which oneend anchors to substrates and the other end provides chemicalfunctionality to the substrate surface.

The existing toolbox for functional modification of material/substratesurfaces includes methods such as self-assembled monolayer (SAM)formation, functionalized silanes, Langmuir-Blodgett deposition,layer-by-layer assembly, and genetically-engineered surface-bindingpeptides. Although widely implemented in research, these conventionalmethods have limitations for widespread practical use. For instance,chemical specificity between interfacial modifiers and substrates (e.g.,alkanethiols on noble metals and silanes on oxides) and complexinstrumentation are typically required. In addition, the substratesize/shape (Langmuir-Blodgett deposition) is often limited, ormulti-step procedures for implementation (layer-by-layer assembly andsurface-binding genetically engineered peptides) are required. Further,existing compounds are often expensive and/or difficult to use.

Methods comprising the single-step coating of substrates with activeagents are known in the art. For example, dopamine is capable ofspontaneously modifying a variety of substrate surfaces under oxidativeconditions (see U.S. Pat. No. 8,541,060 to Messersmith et al.). Dopaminecontains a primary amine that facilitates intramolecular cyclization toform the 5,6-dihydroxyindole intermediate that is essential forpolydopamine formation. Thus, one of skill in the art would have noreasonable expectation that the method disclosed in the '060 patentwould be successful using a nitrogen-free surface modifying agent.Furthermore, polydopamine coatings as described in '060, relatedpatents, and in the academic literature are, without exception, darkcolored coatings, as they are closely related to the chemicalcomposition of melanin pigments. The dark color conferred bypolydopamine coatings is problematic for many practical applications ofthe technology where masking or discoloration of the inherent substrateappearance is to be avoided for aesthetic or performance reasons.

In addition, coatings derived from natural sources are also known to theart. For example, tannic acid has been used to modify substrate surfaces(see Caruso et al. 2013). The Caruso art requires the use of trivalentmetal ions (Fe³⁺, V³⁺, Gd³⁺, or Cr³⁺ ions) and relies on metal-oxygencoordination bonds formed between the tannic acid and trivalent metalions for formation of the coating. They show that their coatings do notform in the absence of trivalent metal ions. Additionally, thesecoordination-based coatings require an acidic-to-basic pH adjustment toform. Furthermore, the Caruso art produces coatings that are darklycolored and are inherently unstable at pH values less than 7.0.Moreover, the coatings based on Caruso art utilize high concentrationsof iron, which will likely be toxic to biological systems if biomedicalapplications are pursued. Coatings that incorporate tannic acid as onecomponent of a multi-component coating have been formed by so-calledlayer-by-layer technology (see Shutava et al. 2005). However,layer-by-layer coatings involve multi-step deposition processes andrequire the use of other molecules for formation. In contrast, thepresent invention describes a general method requiring only aplant-based or plant-inspired phenol or polyphenol precursor compounddeposited in a single step to yield colorless coatings that are stableover a wide range of pH conditions.

Accordingly, cost-effective and easy-to-use compounds and methods forthe surface-independent modification of a substrate whereby specificfunctional moieties can be displayed on the surface are needed.

BRIEF SUMMARY OF THE INVENTION

The inventors demonstrate herein that nitrogen-free phenolic compoundscan be used to form macromolecular coatings on both nonporous and poroussolid substrates. Specifically, under high ionic strength (saline)conditions in the presence of atmospheric oxygen, aqueous solutions ofnitrogen-free phenolic compounds form coatings on immersed substrates.The resultant thin film coatings can be deposited in a single step ontoa wide range of materials of variable composition (e.g., organic andinorganic, including gold, steel, silicone rubber, and Teflon®) andsurface wettability (both hydrophilic and hydrophobic). A wide range ofnitrogen-free phenolic compounds can be used as coating precursors,including without limitation natural polyphenols extracted from planttissue and synthetic phenolics.

The resulting coatings induce little or no discoloration of theunderlying substrate and display intrinsic properties of great practicalsignificance. These include inherent contact—and solution-basedantibacterial properties. The coatings of the invention also haveinherent antioxidant properties, and we demonstrate their use in metalremoval from solvents. Furthermore, the coatings of the invention areeasily functionalized at the macro-, micro-, and nano-scales,facilitating a variety of additional practical uses. For example, theinnate ability of polyphenols to sequester and precipitate polypeptidesand proteins can be exploited in these coatings for the purpose ofimmobilizing biological and synthetic molecules containing nucleophilicsites, such as primary amine or sulfhydryl functionalities. Successfulincorporation of antifouling polymers onto pyrogallol-coated surfacescan be achieved for inhibiting biofouling and through the incorporationof hydrophobic molecules, super-hydrophobic surfaces can be designed.

In another example, the high reduction/oxidation (redox) potential ofthe coatings of the invention permits electroless metallization of noblemetals such as silver through simple immersion of coated substrates intosilver salt solutions. Deposition of the coatings of the invention ontonanoparticles can be utilized for tuning of bulk and surface propertiesof nanoparticles, as we show by tuning the longitudinal plasmonresonance of gold nanorods to preferred wavelengths by deposition of thecoating followed by controlled electroless metallization of silvershells of variable thickness.

Uniting the ability to deposit onto many types of substrates, remarkableversatility in functional properties and little or no discoloration ofthe substrate, the coatings of the invention represent a promisingtechnology for use in a variety of consumer, industrial, military andbiomedical applications.

In a first aspect, the invention encompasses a method of forming acoating on a substrate surface. The method includes the step ofcontacting at least a portion of the substrate surface with an aqueoussolution that includes an effective amount of one or more natural orsynthetic nitrogen-free phenolic compounds, whereby a coating forms onthe substrate surface. In some embodiments, the solution may be a salinesolution.

In some embodiments, the one or more phenolic compounds may includepolyphenols, such as epigallocatechin-3-gallate (EGCG), epigallocatechin(EGC), epicatechin gallate (ECG), or tannic acid. In some embodiments,the one or more phenolic compounds may include other compounds, such asgallic acid and pyrogallol. In some embodiments, the one or morepolyphenols can optionally be extracted from plant materials, such asgreen tea or from cacao beans.

In some embodiments, the solution includes NaCl. In some embodiments,the saline solution is basic. In some such embodiments, the pH of thesolution is about 7.8.

In some embodiments, the substrate surface is gold, titanium dioxide,silica, polycarbonate, polytetrafluoroethylene, polystyrene, titanium,and/or stainless steel.

Some embodiments include the optional additional step of contacting theresulting coating with a reactive moiety, whereby the reactive moietyreacts with and becomes bound to the coating. In some such embodiments,the reactive moiety includes a nucleophile or a metal ion. Optionally,the metal ion can be silver ion.

In some embodiments, the reactive moiety comprises silver ion (Ag⁺), andelemental silver becomes bound to the coating. Optionally, some suchembodiments further include the step of contacting the resulting coatingwith an alkanethiol, whereby the coating becomes superhydrophobic.

In some embodiments, the nucleophile is comprised of a protein or anamine- or thiol-functionalized polymer, including, for example,poly(ethylene glycol) (PEG).

The presence of the amine moiety in dopamine allows for cyclizationunder oxidative conditions (creating the critical dihydroxyindolestructure), yielding enhanced reactivity allowing for furtheroligomerization and polymerization. Such intuition cannot be translatedto phenolic coatings based on absence of amines. Precluded by itsmelanin-like nature, polydopamine coatings cannot be engineer to affordcolorless surface modifications. Phenolic coatings, though, can be tunedto derive a completely colorless material. The phenolic coatings of thepresent invention also exhibit contact-based antioxidant, antibacterial,antifungal, and anti-inflammatory properties, which are not observedusing polydopamine coatings. Additionally, phenolic coatings can betuned to reduce water contact angles into the superhydrophilic regime(less than 10°), a property that can never be achieved usingpolydopamine coatings, which display contact angles of approximately50°.

In a second aspect, the invention encompasses a coating on a surfacesubstrate that is produced by the methods described above.

In a third aspect, the invention encompasses a method of inhibitingbacterial growth on a substrate. The method includes the step ofdepositing a coating on a substrate surface according to any of themethods described above, whereby the coated substrate effectively killsbacteria on contact or inhibits bacterial growth.

In a fourth aspect, the invention encompasses a method of reducing theconcentration of metal in a liquid. The method includes the step ofcontacting the liquid with the coating as described above, whereby atleast some of the metal ions in the sample are captured by the coatingand removed from the liquid.

In some embodiments, the heavy metal cations removed by the method mayoptionally include silver.

In a fifth aspect, the invention encompasses a kit for coating asubstrate surface. The kit includes an effective amount of one or morephenolic compounds, and/or one or more polyphenols, and instructions foruse. Optionally, the kit may further include a salt and a buffer.Optionally, the salt is sodium chloride. Optionally, the kit may furtherinclude a reactive moiety made up of a nucleophile or a metal ion. Themetal ion may optionally be silver.

In a sixth aspect, the invention encompasses a method of forming acoating on a substrate surface. The method includes the step ofcontacting at least a portion of the substrate surface with an aqueoussolution comprising an effective amount of one or more nitrogen-freephenolic compounds, whereby a coating forms on the substrate surface.

In some embodiments, the one or more nitrogen-free phenolic compoundsmay include pyrogallol (PG), epigallocatechin-3-gallate (EGCG),epigallocatechin (EGC), epicatechin gallate (ECG), tannic acid (TA),hydroxyhydroquinone (HHQ), catechin, morin, quercetin, naringenin,naringin, rutin, phloroglucinol, catechol, resorcinol, hydroquinone,phenol, gallic acid, and/or stereoisomers thereof.

As described above, previously developed tannic acid coatings by Carusoet al. are molecular constructs achieved through chemical coordinationbetween galloyl groups of tannic acid and Fe³⁺ (although V³⁺, Gd³⁺, andCr³⁺ were also used). Because coordination bonds are labile in acidicconditions, the tannic acid and Fe³⁺ films degrade rapidly in weaklyacidic conditions. Also, because coordination bonds are the crucialaspect of Caruso's materials, the absence of trivalent metal ionsprevent the formation of any coatings. Further, the actual coatings donot form unless the pH is raised from the initial acidic conditions(acidic due to the Fe³⁺) to pH 7.4-8.0. This change induces theformation of coordination bonds. Further still, Caruso's method providesresulting films that are very dark in appearance.

In contrast, the currently claimed phenolic coating methodology reactswith dissolved oxygen in buffered saline (without the need for a changein pH) to deposit coatings, which appear to contain both covalent C—Cbonds and non-covalent interactions (hydrophobic interactions, pi-pistacking, hydrogen bonds). Our phenolic coatings can be tuned to notdegrade under non-neutral conditions, and, unlike Caruso et al coatings,can be made colorless. Moreover, the lack of heavy metal ions in ourcoatings allows for non-toxic interactions with mammalian cells, animportant property when considering medical translation of thetechnology. To date, Caruso's materials have not been shown to beantioxidant, antimicrobial, and/or anti-inflammatory. Finally, asopposed to the present invention's ability to remove heavy metals fromwater, the films designed by Caruso can be viewed as a source of heavymetals (Fe³⁺, V³⁺, Gd³⁺, and Cr³⁺).

Although coating deposition occurs in the absence of salts, in someembodiments, the aqueous solution includes an effective amount of one ormore salts. By “effective amount” we mean an amount effective to achievean optimal formation of a coating on the substrate surface, where“optimal” is defined as a desired coating thickness or a desired rate ofcoating formation. In some embodiments the presence of the saltincreases the coating thickness and/or rate of coating formation.Although the mechanism of interaction between phenol or polyphenolcoating precursor and salt is not fully understood and likely varies bythe identity of the salt, we anticipate in some cases the optimizationof thickness and/or rate afforded by addition of salt is due to ionicstrength induced intermolecular interactions like charge shieldingbetween molecules, leading to aggregation and deposition of molecules inthe form of a coating. The one or more salts may include a sodium salt,a potassium salt, a calcium salt, a magnesium salt, a copper salt,and/or a zinc salt. In some such embodiments, the one or more salts mayinclude NaCl, NaNO₃, Na₂SO₄, KCl, K₂SO₄, MgCl₂, CaCl₂, CuCl₂, and/orZnCl₂.

In some embodiments, no salt addition is necessary to spontaneouslydeposit a coating. For example, the deposition of pyrogallol coating onnanoparticles was optimally achieved from salt-free solution. However,in other embodiments, at least one of the one or more salts in theaqueous solution is at a concentration of at least 0.001 mM. However, inother embodiments, at least one of the one or more salts in the aqueoussolution is at a concentration of at least 10 mM. In some otherembodiments, at least one of the one or more salts in the aqueoussolution is at a concentration of at least 50 mM. In some embodiments,the salt concentration is optionally between 0 mM and a saturatedsolution, 0.001 mM and 1 mM, between 1 mM and 1,000 mM, between 10 and10,000 mM, between 15 and 10,000 mM, between 20 and 10,000 mM, between30 and 10,000 mM, between 40 and 10,000 mM, between 50 and 10,000 mM,between 10 and 1,000 mM, between 15 and 1,000 mM, between 20 and 1,000mM, between 30 and 1,000 mM, between 40 and 1,000 mM, or between 50 and1,000 mM.

In some embodiments, the aqueous solution may further include a buffer.

In some embodiments, the pH of the aqueous solution is between 3.0 and9.0; more preferably, the pH of the aqueous solution is between 6.0 and9.0.

Some embodiments further include the step of contacting the resultingcoating with a reactive moiety, whereby the reactive moiety reacts withand becomes bound to the coating. Optionally, the reactive moiety mayinclude a nucleophile or a metal ion. Exemplary metal ions that could beused include, without limitation, silver ion. Exemplary nucleophilesinclude, without limitation, biomolecules such as proteins oroligonucleotides, and amine- or thiol-functionalized polymers such asamino- or thiol-terminated poly(ethylene glycol) (PEG).

Some embodiments further include the step of contacting the resultingcoating with an alkanethiol, whereby the coating becomessuperhydrophobic.

In a seventh aspect, the invention encompasses a coating on a substratesurface as produced by the method described above (as a sixth aspect).

In an eighth aspect, the invention encompasses a method of inhibitingbacterial growth on a substrate by depositing an antibacterial coatingon a substrate surface according to the method described above (as asixth aspect), whereby the coated substrate effectively kills bacteriaon contact or inhibits bacterial growth.

In a ninth aspect, the invention encompasses a method of conferringantioxidant properties on a substrate by depositing an antioxidantcoating on a substrate surface according to the method described above(as a sixth aspect), whereby the coated substrate has antioxidantactivity.

In a tenth aspect, the invention encompasses a method of reducinginflammation caused by contact of cells or tissues with a substrate bydepositing an anti-inflammatory coating on a substrate surface accordingto the method described above (as a sixth aspect), whereby the coatedsubstrate has anti-inflammatory activity.

In an eleventh aspect, the invention encompasses a method of removingmetal ions from a liquid. The method includes the step of contacting theliquid with the coating described above (as a seventh aspect), wherebyat least some of the metal ions in the liquid are captured by thecoating and removed from the aqueous sample.

In some embodiments, the metal ions can include silver ions.

In a twelfth aspect, the invention encompasses a kit for coating asubstrate surface. The kit includes an effective amount of one or morephenolic compounds; one or more salts; and instructions for use.

In some embodiments, the kit further includes a buffer. In someembodiments, the kit further includes a reactive moiety containing anucleophile or a metal ion. An exemplary metal ion that could be usedis, without limitation, silver ion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Color changes associated with modification of polycarbonate (PC)substrates with dark chocolate (DC), green tea (GT) and used green tea(used GT), before (−Ag) and after (+Ag) silver nitrate incubations.

FIG. 2A. Chemical structures of naturally-occurring EC found in greentea and chocolate.

FIG. 2B. Chemical structures of naturally-occurring EGC found in greentea and chocolate.

FIG. 2C. Chemical structures of naturally-occurring ECG found in greentea and chocolate.

FIG. 2D. Chemical structures of naturally-occurring EGCC found in greentea and chocolate.

FIG. 3. Color changes associated with the deposition of coatings derivedfrom naturally-occurring green tea and chocolate phenols or polyphenolson polysulfone (PS), before and after metallization of silver.

FIG. 4A. 1,2,3-trihydroxybenzene (pyrogallol, “PG”) chemical structure.

FIG. 4B. Idealized chemical structure of tannic acid (“TA”), with aglucose core containing ten gallate units. The idealized TA structureshown is only one of many compounds identified as TA in the art, withthe number of gallate units per molecule in a commercial TA samplepotentially ranging from 1-15.

FIG. 5. Carbon-to-oxygen ratios for three substrates (PC, PS & PTFE)modified with pyrogallol, as assessed by XPS.

FIG. 6. Pyrogallol (PG) coating thickness growth on gold as a functionof time.

FIG. 7. XPS analysis, including strength of the underlying substratesignal (bars) and carbon-to-oxygen (C/O) ratio (circles), for variousmaterials coated with PG (dark gray) and TA (light gray).

FIG. 8. Color changes associated with the modification of polycarbonatewith pyrogallol and, subsequently, silver nitrate.

FIG. 9A. Scanning electron micrograph of polytetrafluoroethylene (PTFE).

FIG. 9B. Scanning electron micrograph of PTFE modified with PG.

FIG. 9C. Scanning electron micrograph of PTFE modified with PG andsilver nitrate.

FIG. 9D. Scanning electron micrograph of PTFE modified with pyrogalloland silver nitrate shown at a higher magnification.

FIG. 10. XPS survey spectra of polycarbonate modified with pyrogalloland silver nitrate.

FIG. 11A. C1s detailed XPS spectra of a titanium dioxide (TiO₂) surface.

FIG. 11B. C1s detailed XPS spectra of a titanium dioxide (TiO₂) surfacemodified with mPEG-amine.

FIG. 11C. C1s detailed XPS spectra of a titanium dioxide (TiO₂) surfacemodified with mPEG-thiol.

FIG. 11D. C1s detailed XPS spectra of a titanium dioxide (TiO₂) surfacemodified with pyrogallol (PG).

FIG. 11E. C1s detailed XPS spectra of a titanium dioxide (TiO₂) surfacemodified with pyrogallol and mPEG-amine.

FIG. 11F. C1s detailed XPS spectra of a titanium dioxide (TiO₂) surfacemodified with pyrogallol and mPEG-thiol.

FIG. 12. Contact-mediated antibacterial behavior of pyrogallol-coatedpolycarbonate (PC) against Pseudomonas aeruginosa.

FIG. 13A. SEM micrograph of silicon surfaces modified with polystyrenebeads, pyrogallol, silver, and octadecane thiol at high magnification.

FIG. 13B. SEM micrograph of silicon surfaces modified with polystyrenebeads, pyrogallol, silver, and octadecanethiol at low magnification.

FIG. 13C. Image of the modified silicon surfaces with standing waterdroplet on the resultant superhydrophobic surface.

FIG. 14A. Secondary Electron (SE) mode illustrates the diffuse layer,likely attributable to bound pyrogallol, surrounding the gold nanorodscore in STEM micrographs of gold nanorods modified with pyrogallol.

FIG. 14B. Transmission electron (TE) mode illustrates the diffuse layer,likely attributable to bound pyrogallol, surrounding the gold nanorodscore in STEM micrographs of gold nanorods modified with pyrogallol.

FIG. 15. STEM micrograph of a single gold nanorod modified withpyrogallol, obtained using secondary electron (SE) signal.

FIG. 16A. STEM micrographs of gold nanorods modified with pyrogallol.

FIG. 16B. STEM micrographs of gold nanorods modified with pyrogallolfollowed by incorporation of silver.

FIG. 17. EDS elemental composition maps of silver-shell gold-corenanorods formed via pyrogallol modification.

FIG. 18. UV-Vis spectrum sweeps for gold nanorods stabilized withcetyltrimethylammonium bromide (CTAB) and modified with pyrogallol.

FIG. 19A. Gold nanorods plasmon tuning through silver-shellincorporation via pyrogallol. Solutions of gold nanorods modified withpyrogallol, followed by different concentrations of silver nitrate.

FIG. 19B. UV-Vis spectroscopy sweeps for silver-shell gold-corenanorods.

FIG. 20. TA coatings (with and without silver) on various materials.

FIG. 21. EGCG coatings (with and without silver) on various materials.

FIG. 22. Graph showing stability of TA-based coatings.

FIG. 23. Graph showing stability of catechin-based coatings.

FIG. 24. Bar graph showing effect of sodium chloride (NaCl)concentration on PG-based coating thickness. Conditions: 2 mg/mL PG in100 mM bis-Tris at pH 7 with varying amounts of NaCl.

FIG. 25. Graph showing effect of sodium chloride (NaCl) concentrationand pH on PG-based coating thickness. Conditions: 2 mg/mL PG in 100 mMbuffer (bis-Tris or bicine, depending on pH) with varying pH and varyingamounts of NaCl. The numbers at the top of the figure correlate coatingthickness to color.

FIG. 26. Graph showing effect of salt choice on PG-based coatingthickness. Conditions: 2 mg/mL PG in 100 mM bis-Tris at pH 7 with 600 mMsalt. Coating conditions: 2 mg/mL PG in 100 mM bis-Tris with salt at pH7, 8 h. All salts were at a concentration of 600 mM, except CuCl₂ andZnCl₂. These two salts were added at a concentration of 100 μM to 600 mMNaCl.

FIG. 27. Bar graph showing effect of magnesium chloride (MgCl₂)concentration on PG-based coating thickness. Conditions: 2 mg/mL PG in100 mM bis-Tris at pH 7 with varying amounts of MgCl₂.

FIG. 28A. Bare PEEK, and water droplet on bare PEEK.

FIG. 28B. PEEK modified with TA, and water droplet on TA-modified PEEK.

FIG. 28C. PEEK modified with PG, and water droplet on PG-modified PEEK.

FIG. 28D. PEEK modified with Ag⁺, and water droplet on bare PEEKincubated with Ag⁺.

FIG. 28E. PEEK modified with TA and Ag⁺, and water droplet onTA-modified PEEK reacted with Ag⁺.

FIG. 28F. PEEK modified with PG and Ag⁺, and water droplet onPG-modified PEEK reacted with Ag⁺.

FIG. 29A. SEM micrographs of bare PEEK and PEEK modified with Ag⁺ (scalebar is 50 μm).

FIG. 29B. SEM micrographs of TA-modified PEEK with and without Ag⁺(scale bar from A applies to B).

FIG. 29C. SEM micrographs of PG-modified PEEK with and without Ag⁺(scale bar from A applies to C).

FIG. 29D. SEM micrographs of bare PEEK and PEEK modified with Ag (scalebar is 500 nm).

FIG. 29E. SEM micrographs of TA-modified PEEK with and without Ag⁺(scale bar from D applies to E).

FIG. 29F. SEM micrographs of PG-modified PEEK with and without Ag⁺(scale bar from D applies to F).

FIG. 30. Microbial viability on polystyrene (PS), TA-coated PS (PS+TA),and dopamine-coated PS (PS+DA). TA coatings were prepared using a 2mg/mL solution of TA in 100 mM bicine, 600 mM NaCl at pH 7.8. DAcoatings were prepared using a 2 mg/mL solution of DA in 10 mM Tris atpH 8.5. * p<0.05; ** p<0.001. *

FIG. 31. Visualization of polycarbonate samples coated with catechin(Ctn) and epicatechin (ECtn).

FIG. 32: Silver concentration of solutions before and after exposure tobare and phenolic-modified gauze (statistical significance of p<0.05between gauze and stock solution, and p<0.001 for all other comparisonsvia one-way ANOVA).

FIG. 33: Atomic percentage increase in the nitrogen content of TiO₂,TiO₂+PG, and TiO₂+TA surfaces modified with lysozyme in water orbuffered saline.

FIG. 34: Production of reactive oxygen species (ROS) in NIH 3T3fibroblasts cultured on PG- and TA-modified surfaces as a fraction ofbare polystyrene control.

FIG. 35: TNF-α expression by RAW 264.7 murine monocytes on PC, PC+PG andPC+TA surfaces before and after stimulation with LPS.

DETAILED DESCRIPTION OF THE INVENTION

In the specification and in the claims, the terms “including” and“comprising” are open-ended terms and should be interpreted to mean“including, but not limited to . . . .” These terms encompass the morerestrictive terms “consisting essentially of” and “consisting of.”

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, “characterized by” and “having” can beused interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications and patentsspecifically mentioned herein are incorporated by reference in theirentirety for all purposes including describing and disclosing thechemicals, instruments, statistical analyses and methodologies which arereported in the publications which might be used in connection with theinvention. All references cited in this specification are to be taken asindicative of the level of skill in the art. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

The Invention.

The present invention provides a coating formed when an effective amountof an aqueous solution and one or more nitrogen-free phenolic compoundsis applied to a portion of a substrate, wherein the substrate surface iscoated with and modified by the one or more nitrogen-free phenoliccompounds. In one embodiment, the solution may comprise an effectiveamount of one or more salts sufficient to achieve an optimal coating.Methods of making and using the coating, as well as kits for using thecoating, are also provided.

“Phenolic compounds” or “phenols” are nitrogen-free compounds comprisingone or more aromatic rings having at least one hydroxyl group attachedthereto. The one or more aromatic rings are not limited to having asingle hydroxyl group, as in phenol, but may each include more than onehydroxyl group, as in a catechol.

Polyphenols are a structural class of nitrogen-free compoundscharacterized by the presence of two or more phenolic structural units,and may include any number of natural or synthetic precursors that yieldcoatings. The “polyphenols” of the present invention are nitrogen-freeand may be considered either “phenols” or “polyphenols” according to thewidely accepted definitions known to one of skill in the art. Althoughthe chemical “phenol” itself has only one phenolic hydroxyl group on thearomatic ring, a “phenolic structural unit” may include two or morehydroxyl groups on the same aromatic ring. For example, catechol andresorcinol, which include two hydroxyl groups on the same aromatic ring,and pyrogallol and phloroglucinol, which have three hydroxyl groups onthe same aromatic ring, are considered to have a “phenolic structuralunit.” Polyphenols and phenolic structural units may have oxygen-basedsubstituents other than hydroxyl groups; as might be expected, ether andester linkages are common, as are various carboxylic acid derivatives.

Polyphenols are known to modulate gene expression and have been used inthe treatment of cancer. In addition, polyphenols have antioxidantactivity against reactive oxygen species (ROS), which are involved inimmune responses.

The coatings of the invention can be deposited on a substrate surface asa thin (i.e., a monolayer ranging from about 0.5 to about 50 nm or more)coating on virtually any material. Compared to other compounds used tocoat substrates, polyphenols formed from the nitrogen-free phenoliccompounds have the advantage of being inexpensive, adherent, and simpleto deposit onto substrates without the need for surface pre-treatment.Polyphenol nanolayers can form on virtually any material surface,including noble metals, oxides, semiconductors, ceramics, syntheticpolymers, and graphene oxide, as well as on superhydrophobic surfaces.

Polyphenols are found in virtually all families of plants, and cancomprise up to 50% of the dry weight of plant tissue. Polyphenols can bederived naturally (i.e., by extracting them from plants) orsynthetically. By “plant polyphenols”, we mean polyphenols derived fromplant tissues, such as, for example and without limitation, tea leaves,cacao/chocolate, grapes/red wine, etc. In some embodiments, the plantpolyphenols used in the present invention includeepigallocatechin-3-gallate (EGCG), epigallocatechin (EGC) andepicatechin gallate (ECG), and tannic acid. Tannic acid (“TA”), as iswidely understood in the art, includes without limitation a number ofcompounds containing from 1-15 gallic acids bound together with esterlinkages surrounding a glucose core. Commercial TA typically consists ofa mixture of tannic acid molecules of variable molecular weight.Pentagalloyl glucose fits the definition of commercial tannic acid andis, therefore, embodied within the present invention. The coatingprecursor embodied within this invention includes both purifiedcompounds as well as mixtures of multiple compounds derived from planttissue as is found in commercial TA sources. Other nitrogen-freephenolic compounds, such as gallic acid and pyrogallol, can also be usedin the present invention.

By “tea”, we mean any tea leaf containing an effective amount ofpolyphenols. In some embodiments, the tea may be green tea, white tea,red tea, oolong tea, black tea and more.

By “cacao” we mean any cacao plant tissue containing an effective amountof polyphenols. For instance, this includes cacao bean, which contains ahigh concentration of polyphenol. Additionally, cacao liquor is a majorcomponent of chocolate, and includes an effective amount of polyphenol.By “effective amount” we mean an amount of polyphenol sufficient toachieve the optimal coating in the present invention. In someembodiments, the polyphenol may be derived from chocolate, such as darkchocolate having at least 80-90% cacao.

By “tannic acid” we mean a specific form of tannin, a type ofpolyphenol. Commercial tannic acid is usually extracted from Tara pods,gallnuts or Sicilian Sumac leaves and is typically a mixture of manycompounds as described above, any one of which, or any combination ofwhich, can be used to achieve the coatings described in this invention.

By “gallic acid” we mean the crystalline organic acid found in gallnuts,sumach, tea leaves, oak bark, and many other plants, or synthesizedchemically, both in its free state and as part of tannic acid and otherhydrolyzable tannins. Gallic acid is obtained by the hydrolysis oftannic acid. When heated above 220° C., gallic acid loses carbon dioxideto form pyrogallol, or 1,2,3-trihydroxybenzene, C₆H₃(OH)₃.

By “pyrogallol” we mean 1,2,3-trihydroxybenzene, C₆H₃(OH)₃. Pyrogallolcan be produced from gallic acid as noted above and can also beextracted from the aquatic plant Myriophyllum spicatum.

Plant-Derived Polyphenol Coatings.

In one embodiment, the invention comprises a coating formed when asaline solution comprising one or more nitrogen-free phenolic compoundsis applied to a portion of a substrate, wherein the substrate surface iscoated. In some embodiments, the phenolic compounds are derived from aplant derived extract that may include plant polyphenols, such asepigallocatechin-3-gallate (EGCG), epigallocatechin (EGC) andepicatechin gallate (ECG), and tannic acid. Other plant derived nitrogenfree phenolic compounds, including without limitation gallic acid andpyrogallol, can be used.

The coating may also comprise a reactive moiety, wherein the reactivemoiety reacts with and is bound to the coated surface of the substrate.The reactive moiety may comprise a nucleophile or a metal ion, such assilver ion. In some embodiments, a metal salt, such as NaCl, is added toenhance the coating.

By “effective amount” we mean an amount sufficient that, when contactedwith a substrate surface under reactive conditions, is sufficient toeffect the expected reaction. In one embodiment, we mean an amountsufficient to deposit a coating on the substrate.

By “coating” we mean depositing a macromolecular coating on thesubstrate surface. The thickness of the coating may vary according tothe needs of the user and the surface to be treated. Using techniquesknown to one of skill in the art, these variables can be addressedwithout undue experimentation.

By “applied” we mean any method of coating a surface known to the art,including spin-coating, painting, dipping, washing, spraying, brushing,and the like.

By “substrate” we mean any material known to the art, including asurface formed from polycarbonate (PC), polysulfone (PS), polyisoprene(PI), polytetrafluoroethylene (PTFE), silicone rubber (SiR), titaniumdioxide (TiO₂), gold (Au), and aluminum oxide (Al₂O₃). In otherembodiments, the substrate may be any surface formed from polyurethaneand its copolymers, silicone and its copolymers, ethylene vinyl-acetate,thermoplastic elastomers, polyvinyl chloride, polyolefins, cellulosics,polyamides, polytetrafluoroethylenes, polyesters, polycarbonates,polysulfones, and acrylonitrile butadiene styrene copolymers; acrylics;metals and alloys based on titanium, stainless steel, nickel-chrome,nitinol or cobalt-chrome; ceramics of alumina and glass-ceramics and thelike. For instance, the surface can comprise a metallic material or analloy such as, but not limited to, cobalt, nickel, chromium, molybdenum,stainless steel (316L), high nitrogen stainless steel, cobalt chrome,tantalum, nickel-titanium alloy, platinum-iridium alloy, gold,magnesium, or combinations thereof. Surfaces comprising bioabsorbable orbiostable polymers could also be used with the embodiments of thepresent invention.

The substrate may be without limitation a nonporous, porous, membranousor fibrous substrate of any geometry.

The substrate may be a medical device or any part of a medical devicethat comes in contact with a patient, including but not limited toself-expandable stents, balloon-expandable stents, stent-grafts, grafts(e.g., aortic grafts), vascular grafts, artificial heart valves,cerebrospinal fluid shunts, pacemaker electrodes, guide wires,ventricular assist devices, artificial hearts, cardiopulmonary by-passpumps and circuits, blood oxygenators, endocardial leads, catheters,implantable vascular access ports, blood storage bags, vascular stents,blood tubing, central venous catheters, arterial catheters, vasculargrafts, intraaortic balloon pumps, heart valves, cardiovascular sutures,total artificial heart and ventricular assist pumps, blood oxygenators,blood filters, hemodialysis units, hemoperfusion units, plasmapheresisunits, and hybrid artificial organs such as pancreas or liver andartificial lungs and the like.

In alternate embodiments, the invention comprises a method of modifyinga substrate surface comprising contacting at least a portion of thesubstrate with a solution containing an effective amount ofplant-derived polyphenol, wherein the substrate surface is modified. Themethod may also comprise contacting the surface-modified substrate witha reactive moiety, wherein the reactive moiety reacts with and is boundto the modified surface.

Methods of Use.

The coating and methods of the present invention will be useful inimportant fields including biocompatible coatings of medical devices,surface modifications of drug delivery carriers and tissue engineeringscaffolds, biosensors, biofouling-resistant, industrial and consumercoatings, semiconductors, metal removal, control of wetting propertiesof solid or porous objects, application of catalysts to surfaces, andfabrication of next generation electronic displays.

Kits.

In an alternate embodiment of the invention, a kit for preparing andusing the novel macromolecular coating of the present invention isprovided. In one embodiment, the kit comprises a nitrogen-free phenoliccompound according to the present invention and instructions for use.

By “instructions for use” we mean a publication, a recording, a diagram,or any other medium of expression which is used to communicate theusefulness of the invention for one of the purposes set forth herein.The instructional material of the kit can, for example, be affixed to acontainer which contains the present invention or be shipped togetherwith a container which contains the invention. Alternatively, theinstructional material can be shipped separately from the container orprovided on an electronically accessible form on an internet websitewith the intention that the instructional material and the plant-derivedpolyphenol coating be used cooperatively by the recipient.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed description of the present invention is to beregarded as illustrative in nature and not restrictive.

THE EXAMPLES

The following examples are, of course, offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and the followingexamples and fall within the scope of the appended claims.

Example 1 Surface Modification Through Polyphenols From Green Tea andDark Chocolate Extracts Under Mildly-Basic, Saline, Aqueous Conditions

Crude extracts of green tea (GT) and dark chocolate (DC) were preparedfrom raw food products (Table 1) using methods known to the art. Theresultant extracts, in solutions buffered at pH 7.8 with 100 mM bicine,further supplemented with 600 mM NaCl, resulted in adherent coatingdepositions on polycarbonate substrates following 4 h exposure at roomtemperature with mild agitation. The resultant coatings were confirmedthrough X-ray photoelectron spectroscopy, as seen by alteredcarbon-to-oxygen (C/O) ratios. Additionally, the resultant materials,once exposed to 100 mM AgNO₃ for 24 h, were able to metallize silver.

TABLE 1 Elemental composition of polycarbonate (PC) modified with darkchocolate (DC), green tea (GT), and used green tea (used GT) extract,followed by incubation in silver nitrate (Ag), as determined by XPS.Elemental Composition Surface C O N Ag PC 85.3% 14.7% 0.0% 0.0% + Ag84.8% 14.9% 0.0% 0.4% PC + DC 80.6% 17.8% 1.6% 0.0% + Ag 72.0% 19.3%1.4% 7.3% PC + GT 76.8% 22.4% 0.7% 0.0% + Ag 77.3% 18.1% 1.0% 3.6% PC +used GT 74.1% 25.4% 0.6% 0.0% + Ag 71.7% 22.9% 0.7% 4.7%

Aside from XPS analysis, the emergence of a dark amber color wasassociated with metallization of silver onto polycarbonate substratescoated with the polyphenol extracts of the present invention (FIG. 1).The redox properties of the polyphenol coatings were attributed to thepresence of polyphenols, which, in solution, exhibits reductiveproperties towards silver nitrate salts.

Extractions of polyphenol from raw foods were performed as follows:Green tea leaves were combined with 70% methanol in water at aconcentration of 1 g tea leaves per 10 mL solvent. The tea leaves weresonicated for 10 minutes, followed by centrifugation at 1,000×g for 10minutes. The supernatant was saved and the tea leaves were re-subjectedto the same extraction protocol for 2 additional times. Combinedsupernatants were concentrated under reduced pressure, filtered througha 0.22 μm filter, and the resultant fraction frozen and lyophilizedprior to use.

Dark chocolate (90% cacao content) was defatted by sonication of 1 g per10 mL of hexane for 10 minutes, followed by centrifugation at 1,000×gfor 10 minute. The defatting protocol was repeated once more. Thedefatted chocolate was suspended in 70% acetone and 0.2% acetic acid inwater at a concentration of 1 g chocolate per 10 mL solvent. Thechocolate was sonicated for 10 minutes, followed by centrifugation at1,000×g for 10 minutes. The supernatant was saved and the chocolate wasre-subjected to the same extraction protocol for 2 additional times.Combined supernatants were concentrated under reduced pressure, filteredthrough a 0.22 μm filter, and the resultant fraction frozen andlyophilized prior to use.

Used green tea was obtained after brewing a cup of green tea for 10minutes. The remaining green tea leaves were removed from the brewedliquid and combined with 0.12 M HCl at a concentration of 1 g tea leavesper 10 mL solvent. The tea leaves were sonicated for 10 minutes,followed by centrifugation at 1,000×g for 10 minutes. The supernatantwas filtered through a 0.22 μm filter, frozen and lyophilized prior touse.

Given the prevalence of various trihydroxybenzene-substituted polyphenolmolecules, such as epigallocatechin-3-gallate (EGCG), epigallocatechin(EGC) and epicatechin gallate (ECG), previously identified in green teaand dark chocolate, specific purified compounds were utilized to depositpolyphenol-based coatings (FIG. 2). Qualitative observations of surfacemodified with EGCG, EGC and ECG, following metallization in silvernitrate solution, indicated that coatings are deposited as expected(FIG. 3).

Example 2 Pyrogallol Coating Deposition Under Mildly-Basic, Saline,Aqueous Conditions

Pyrogallol (PG, FIG. 4) coating deposition under mildly-basic, saline,aqueous condition was achieved on a variety of substrates. Pyrogallol isa more cost-effective phenolic compound to use and obtain, as comparedto many other phenols and polyphenols. The dip coating strategy wasperformed by incubating the choice substrate in a 0.1 to 2.0 mg/mLsolution of pyrogallol, buffered at pH 7.8 with 100 mM bicine, furthersupplemented with 600 mM NaCl to adjust ionic strength. The incubationwas performed at room temperature with mild agitation for duration of0.5 h to 24 h.

To date, substrates made of the following materials have beensuccessfully modified with Pyrogallol according to the presentinvention: Polycarbonate (PC), Polysulfone (PS), Polyisoprene (PI),Polytetrafluoroethylene (PTFE), Poly(ether ether ketone) (PEEK),Polystyrene, Silicone rubber (SiR), Titanium dioxide (TiO₂), Gold (Au),and Aluminum oxide (Al₂O₃). We expect the pyrogallol coating of thepresent invention to be effective on all substrates.

Modification of the various substrates has been confirmed by X-rayphotoelectron spectroscopy (XPS) and contact angle measurement, usingwater as solvent. XPS data suggests that the theoreticalcarbon-to-oxygen ration (C/O) of 2:1 is approached after substrates havebeen incubated with pyrogallol (FIG. 5), and the measured static contactangles readily decreased to approximately 20° after pyrogallolmodification.

Using gold substrates, kinetic studies of coating thickness as afunction of pyrogallol concentration, ionic strength adjustment and timehave been performed (FIG. 6). Approximately 18 nm-thick coatings wereachieved using 2 mg/mL pyrogallol solutions after 15 h. The use ofNaCl-supplementation was found to enhance coating thickness.Additionally, we have found that tannic acid (TA, FIG. 4) can bedeposited on a variety of substrates under identical incubationconditions (FIG. 7).

Example 3 Electroless Metallization of Silver on Surfaces via Pyrogallol

Surfaces modified with pyrogallol are capable of reducing ionic silver(Ag⁺) from aqueous silver nitrate solutions. By “electroless”, we meanan otherwise spontaneous process not requiring the use of an appliedelectric field to induce metallic coating formation. As-preparedsubstrates coated with pyrogallol are immersed in 100 mM AgNO₃ for 24 atroom temperature with mild agitation. Upon visual inspection,polycarbonate substrates modified with pyrogallol and AgNO₃ appear darkamber in color (FIG. 8). Through scanning electron microscopy (SEM),metallic nanoparticle formation is observed on polytetrafluoroethylenesubstrates modified with pyrogallol and AgNO₃ (FIG. 9). Surfacecompositional analysis through XPS indicated incorporation of silverinto the coating (FIG. 10 and Table 2), as seen by the emergence of Ag3dpeaks.

TABLE 2 Elemental composition of polycarbonate (PC) modified withpyrogallol (PG) and silver nitrate (Ag), as determined by XPS. ElementalComposition Surface C1s O1s N1s Ag3d PC 85.3% 14.7% 0.0% 0.0% PC + PG67.4% 32.2% 0.4% 0.0% PC + PG + Ag 52.9% 26.7% 0.5% 19.9%

Substrates modified with pyrogallol were successfully functionalizedwith methoxy poly(ethylene glycol)-thiol (mPEG-SH) and methoxypoly(ethylene glycol)-amine (mPEG-NH₂). Following modification withpyrogallol, choice substrates were incubated in 1 mM mPEG-SH andmPEG-NH₂ solutions (5k MW mPEG used), buffered at pH 8.5 with 10 mMbicine, for 10 minutes at room temperature with mild agitation. XPSanalysis indicates that the emergence of C1s signal (286.7 eV)associated with C—O bonds occurs following incubation with mPEG inbuffer (FIG. 11). Non-buffered solutions do not yield an appreciablechange in the detailed C1s spectrum, relative to the unmodifiedpolycarbonate control.

Additionally, we were able to perform co-incubations of pyrogallol withan 8-arm poly(ethylene glycol)-amine (8-arm PEG-amine), resulting inPEGylated coatings on polycarbonate. Pyrogallol and 8-arm PEG-amine (20k MW) were combined in a 1:1 or 10:1 ratio of pyrogallol:amine group inan aqueous solution buffered at pH 7.8 with bicine, with or without 600mM NaCl, for 18 h with shaking under room temperature. XPS analysisindicates a similar trend that has been seen when grafting mPEG-amine toa pyrogallol -modified substrate. 1:1 pyrogallol:amine with salt appearsto result in the greatest PEG signature, as assessed by C1s signal, whencompared to salt-free and 10:1 pyrogallol:amine conditions.Additionally, a nitrogen signal is detected for 1:1 pyrogallol:amineconditions using salt in the incubation solution (Table 3).

TABLE 3 Elemental composition of polycarbonate (PC) modified withpyrogallol (PG) and 8-arm PEG-amine, as determined by XPS. ElementalComposition Surface C O N PC 85.3% 14.7% 0.0% PC + 1:1 PG:amine − NaCl77.7% 22.3% 0.0% PC + 1:1 PG:amine + NaCl 73.1% 25.4% 1.3% PC + 10:1PG:amine − NaCl 79.3% 20.7% 0.0% PC + 10:1 PG:amine + NaCl 76.5% 23.3%0.2%

The final concentrations were: 1:1 pyrogallol:amine—0.1 mg/mL pyrogalloland 2.0 mg/mL 8-arm PEG-amine and 10:1 pyrogallol:amine—1.0 mg/mLpyrogallol and 2.0 mg/mL 8-arm PEG-amine.

Subsequent exposure of mPEG-SH modified substrates to Pseudomonasaeruginosa (ATCC 7700) resulted in abrogated attachment of bacteria whencompared to unmodified controls. The assay was performed by exposing thedesired substrates to bacteria at 10⁸ CFU/mL in 0.85% NaCl solution for24 h under static conditions. Following exposure, the resultant sampleswere rinsed, stained for bacteria, and the area coverage determinedthrough fluorescence microscopy.

Example 4 Contact-Mediated Antibacterial Properties ofPyrogallol-Modified Surfaces

Polycarbonate substrates modified with 2 mg/mL pyrogallol for 8 h wereexposed to Pseudomonas aeruginosa and Staphylococcus aureus for 24 h.Under the experimental conditions used, the Pseudomonas aeruginosabacteria were in direct contact with the pyrogallol-modified surface(FIG. 12). Viability of the cells was assessed by using a live/deadstain (Invitrogen). A similar experiment was performed while maintainingcells away from the pyrogallol-modified surface, with no marked increasein antibacterial behavior observed relative to unmodified controlsurfaces. Based on the antibacterial data, it appears that theantibacterial properties of the pyrogallol-modified surfaces arecontact-mediated.

Example 5 Superhydrophobic Surface Modifications Enabled ThroughPyrogallol Coating

Superhydrophobic surfaces were generated as follows: Polystyrene beads(1 μm diameter) were deposited onto silicon wafer substrates withspin-coating. Pyrogallol was deposited onto the substrate surface aspreviously described. Silver nanoparticles were nucleated onto theentire pyrogallol-modified surface from aqueous silver nitrate solution.Alkanethiol solutions (5 mM octadecanethiol and 5 mM dodecanethiol inethanol) were used to graft hydrophobic molecules to the resultantsurfaces.

As assessed by scanning electron microscopy (SEM), the silicon surfacesmodified with polystyrene beads, pyrogallol, silver nitrate, andalkanethiol indicate the presence of a hierarchical structure of micron-and nano-scale features, in an attempt to mimic the structure of thelotus leaf (FIG. 13). The measured contact angles exceed 150 degrees,classifying the resultant surface as superhydrophobic.

Example 6 Modification of Gold Nanorods with Pyrogallol and SubsequentPlasmon Tuning by Silver-Shell Formation

Gold nanorods, prepared by cetyltrimethylammonium bromide(CTAB)-templated synthesis, were modified with pyrogallol andsubsequently metallized with a silver shell. The protocol forpreparation of pyrogallol-coated gold nanorods, additionally modifiedwith a silver shell, is as follows:

As-prepared CTAB-stabilized gold nanorods were centrifuged at 9,000relative centrifugal force (rcf) for 10 minutes and the supernatant wasdiscarded. The gold nanorod pellet was resuspended in 0.1 mg/mLpyrogallol buffered at pH 7.8 with 100 mM bicine without the addition ofa metal salt, and sonicated for 20 minutes. For silver shell formation,0-4,000 μM of AgNO₃ was added to the reaction mixture and the solutionwas sonicated for an additional 10 min.

Prior to centrifugation, the pyrogallol coating reaction is terminatedby addition of concentrated acetic acid in a 1:1 molar ratio withbicine. Following centrifugation, the pyrogallol-coated gold nanorodsare resuspended in water.

The pyrogallol-based layer was visible under secondary electron (SE)mode using scanning transmission electron microscopy (STEM) (FIG. 14),measuring approximately 5 nm in thickness (FIG. 15). The pyrogallollayer appears to stabilize the gold nanorods in solution without theneed for an additional surfactant, like CTAB. The pyrogallol coating wasutilized to deposit metallic silver on the gold nanorods surface. Asseen by Z-contrast images through STEM, the less electron-dense silvermetal surrounds the more electron-dense gold nanorods core (FIG. 16).Energy-dispersive electron spectroscopy (EDS) was utilized to confirmthe silver-shell gold-core identity of the pyrogallol-modified goldnanorods constructs (FIG. 17).

While the use of pyrogallol coatings alone does not dramatically alterthe spectroscopic properties of gold nanorods (FIG. 18), the addition ofa silver-shell can be used to tune the longitudinal plasmon wavelengthfor desired applications (FIG. 19). The thickness of the silver shellwas influenced by the concentration of silver nitrate used duringmetallization (FIG. 17). Such plasmon wavelength tunability isattractive for designing metal nanorods excitable at specificwavelengths, for example, to match the emission properties of anavailable light source.

Example 7 Coating Ability at pH 3, 4, 5, 6, 7, 8, and 9

The ability to form coatings over a wide range of pH (3-9) values wasconfirmed. As an example, tannic acid was dissolved at a concentrationof 1 mg/mL in solutions of 100 mM buffer and 600 mM NaCl at pH 7(bis-Tris), 8 (bicine), and 9 (bicine). Pieces of titanium dioxide(TiO₂) were submerged in the tannic acid solutions, rocked for 24 h,rinsed thoroughly with water, and dried with N₂ gas. The coating abilitywas assessed via x-ray photoelectron spectroscopy (XPS) by monitoringthe degree to which the Ti2p signal—corresponding to the underlyingsubstrate (TiO₂)—was diminished (Table 4). If the best coatingperformance occurred at pH 7, a second round of coatings were conductedat pH 3 (formate), 4 (formate), 5 (acetate), and 6 (bis-Tris). Thecoating performance was assessed identically to what was describedabove.

The interpretation of the data is straightforward and based uponphotoelectron escape from the sample. As a coating is deposited on topof the substrate, the photoelectrons representing the substrate areattenuated. Therefore the lower the substrate signal, the better themolecule was able to form a coating. Coatings greater than approximately10 nm in thickness entirely eliminate the signal from the substratebecause the photoelectrons cannot escape and be detected. In Table 4, avalue of 100% indicates no coating formation, and a value of 0%indicates at least 10 nm of coating.

Using this strategy, tannic acid, epigallocatechin gallate (EGCG),pyrogallol (PG), hydroxyhydroquinone (HHQ), gallic acid, ellagic acid,catechin, epigallocatechin (EGC), morin, quercetin, naringenin,naringin, rutin, phloroglucinol, catechol, resorcinol, hydroquinone,phenol, and resveratrol were investigated. Tannic acid, EGCG, PG, HHQ,catechin, EGC, morin, quercetin, naringenin, naringin, rutin,phloroglucinol, catechol, resorcinol, hydroquinone, and phenol (allmolecules other than gallic acid, ellagic acid, resorcinol, andresveratrol) were found to be capable of forming coatings between pH 3and 9.

TABLE 4 Ti2p signal strength after coating TiO₂ sample with candidatemolecules. Ti2p Signal Strength (%) Molecule pH 3 pH 4 pH 5 pH 6 pH 7 pH8 pH 9 Gallic acid ^(d) ^(d) ^(d) ^(d) 94.0 95.0 88.3 Tannic acid 65.256.8 62.9  0.0  0.0 62.9 72.7 Ellagic acid ^(d) ^(d) ^(d) ^(d) ^(c) ^(c)^(c) Catechin ^(d) ^(d) ^(d) ^(d) 44.9 49.0  0.0 EGC ^(d) ^(d) ^(d) ^(d)90.2 84.1 41.2 (0.1 mg/mL) EGCG 81.5 87.4 82.5 52.4  8.7 43.8 91.8 (0.1mg/mL) EGCG 70.1 71.5 71.8 67.7  0.3 45.5 83.8 Morin ^(d) ^(d) ^(d) ^(d)^(c) 45.9 54.7 Quercetin ^(d) ^(d) ^(d) ^(d) ^(c) ^(c) 73.3 Naringenin^(d) ^(d) ^(d) ^(d) ^(c) 80.2 90.1 Naringin ^(d) ^(d) ^(d) ^(d) 90.979.0 80.0 Rutin ^(d) ^(d) ^(d) ^(d) ^(c) 65.1 88.0 Phloroglucinol ^(d)^(d) ^(d) ^(d) 98.0 82.8 77.4 PG 100.9  96.6 87.6 35.3 11.0 58.0 86.9Catechol ^(d) ^(d) ^(d) ^(d) 51.7  8.5 81.1 Resorcinol ^(d) ^(d) ^(d)^(d) 94.7 85.0 90.9 Hydroquinone ^(d) ^(d) ^(d) ^(d) 89.3 89.6 70.6 HHQ18.6 32.9 22.0  1.1 51.6 88.2 76.8 Phenol ^(d) ^(d) ^(d) ^(d) 102.2 68.5 79.0 Resveratrol ^(d) ^(d) ^(d) ^(d) ^(c) 97.7 95.3 (0.5 mg/mL)Buffer Controls 86.5 85.3 91.7 90.7 100   97.3 92.8 ^(a) All coatingprecursors were dissolved at 1 mg/mL unless otherwise indicated. ^(b)Values associated with successful coatings (at least 20% signalreduction) are in bold. ^(c) Represents insolubility of the precursor.^(d) Represents conditions that were not investigated (explained inExample 7).

In order to further corroborate XPS results, select molecules werefurther studied via ellipsometry for their ability to form coatings.Compounds were tested at their optimal pH conditions, which were definedas the pH that caused the greatest signal attenuation in the XPS trials(represented in Table 1). As an example, tannic acid was dissolved at aconcentration of 1 mg/mL in solutions of 100 mM bis-Tris and 600 mM NaClat pH 7. Three TiO₂ samples were submerged in the tannic acid solutions,rocked for 24 h, rinsed thoroughly with water, and dried with N₂ gas.Coating thickness was then measured by ellipsometry (Table 5).

Following a similar procedure, tannic acid, pyrogallol, catechol,catechin, HHQ, EGCG, and morin were coated at pH 6, 7, 8, 9, 6, 7, and8, respectively. Tannic acid was tested at pH 6 and 7 because both ledto 100% Ti2p signal reduction in Example 7. Coating thicknesses weredetermined via ellipsometry, as explained above.

When phenolic compounds were omitted (buffer controls), no coating wasobserved at any pH.

TABLE 5 Coating thickness (nm) on TiO₂ and polycarbonate as determinedby ellipsometry. Substrate Coating Precursor Conditions CoatingThickness (nm) TiO₂ Tannic acid 24 h, pH 6 5.8 ± 0.9 Tannic acid 24 h,pH 7 109.3 ± 7.1  Pyrogallol 24 h, pH 7 19.0 ± 1.9  Catechol 48 h, pH 85.0 ± 0.1 Catechin 48 h, pH 9 250.3 ± 51.8  Hydroxyhydroquinone 48 h, pH6 48.8 ± 10.5 Epigallocatechin 48 h, pH 7 34.3 ± 7.2  gallate Morin 48h, pH 8 1.9 ± 0.2

Example 8 Determination of Coloration Caused by Coating

In order to determine the discoloration caused by phenolic coatings,several materials were modified with phenolic films. Representativeexamples were chosen from metals, metal oxides, organic polymers,inorganic polymers, and bulk materials. Substrates include TiO₂, silicawafer with a thermal oxide layer (SiO₂), gold (Au), polycarbonate (PC),polytetrafluoroethylene (PTFE), polystyrene (PS), nylon 6-12, poly(etherether ketone) (PEEK), poly(p-phenylene sulfide) (PPS), and stainlesssteel (SS). As an example, tannic acid was dissolved at a concentrationof 1 mg/mL in solutions of 100 mM bis-Tris and 600 mM NaCl at pH 7.Samples were submerged in the tannic acid solutions, rocked for 24 h,rinsed thoroughly with water, and dried with N₂ gas. Coatings wererevealed by reacting with silver nitrate (Ag⁺) and recorded visuallythrough digital photography (FIG. 20).

Following a similar procedure, various materials were coated with PG atpH 7, catechol at pH 8, catechin at pH 9, HHQ at pH 6, and EGCG at pH 7.Representative results are shown in FIG. 21 for coatings derived fromEGCG.

Example 9 Substrate-Independent Coatings

In order to determine the ability of coatings to form, independent ofthe substrate composition, several materials were investigated.Representative examples were chosen from metals, metal oxides, organicpolymers, inorganic polymers, and bulk materials. Substrates includeTiO₂, silica wafer with a thermal oxide layer (SiO₂), gold (Au),polycarbonate (PC), polytetrafluoroethylene (PTFE), polystyrene (PS),nylon 6-12, poly(ether ether ketone) (PEEK), poly(p-phenylene sulfide)(PPS), and stainless steel (SS). Tannic acid was dissolved at aconcentration of 1 mg/mL in solutions of 100 mM bis-Tris and 600 mM NaClat pH 7. Samples were submerged in the tannic acid solutions, rocked for24 h, rinsed thoroughly with water, and dried with N₂ gas. The presenceof a coating was determined by XPS and by silver deposition (Table 6).

Following a similar procedure, various materials were coated with PG,catechol, catechin, HHQ, and EGCG at pH 7, 8, 9, 6, and 7, respectively.Coating ability was determined by XPS, ellipsometry, and silverdeposition. Notably, although the tested compounds coated a wide varietyof materials, none of the compounds tested were capable of coating asilica wafer (or glass).

TABLE 6 Coating ability on a wide range of substrate materials. CoatingCoating Formed? Precursor TiO₂ SiO₂ Au PC PTFE PS Nylon PEEK PPS SS TAYes No Yes Yes Yes Yes Yes Yes Yes Yes PG Yes No Yes Yes Yes Yes Yes YesYes Yes Catechol Yes No Yes Yes Yes Yes Yes Yes Yes Yes Catechin Yes NoYes Yes Yes Yes Yes Yes Yes Yes HHQ Yes No Yes Yes Yes Yes Yes Yes YesYes EGCG Yes No Yes Yes Yes Yes Yes Yes Yes Yes ^(a) The presence of acoating was confirmed by XPS, ellipsometry, and silver deposition, asdescribed in Example 7. ^(b) TiO₂—titanium dioxide; SiO₂—silica;Au—gold; PC—polycarbonate; PTFE—polytetrafluoroethylene; PS—polystyrene;Nylon—nylon 6-12; PEEK—poly(ether ether ketone); PPS—poly(p-phenylenesulfide); SS—stainless steel.

Example 10 Relating pK_(a) to Coating pH

In order to investigate the relationship between coating pH anddeprotonation, coating precursor molecules were studied viapotentiometric titrations. A solution of tannic acid was made bydissolving ˜1 mg in 2 mL of 100 mM KCl in a glass test tube to performthe titration. The TA and KOH solutions were degassed by aggressivelysparging argon for no less than 30 min. Base was titrated in 1.0 μLincrements, and the total amount added was 250 μL. If oxygen is purgedcorrectly, the phenolic solution should display little or no change incolor throughout the titration. The first pK_(a) was calculated byinitially plotting the volume of base added versus pH. The firstderivative of this curve was then plotted as a function of pH, and thepK_(a) was calculated by determining the pH associated with any localmaxima.

Following a similar procedure, the first pK_(a) values for PG, catechol,catechin, HHQ, and EGCG were determined via potentiometric titration(Table 7).

TABLE 7 pK_(a) and optimal coating pH values associated with coatingprecursors. Molecule First pK_(a) Optimal Coating pH pK_(a) − pHCatechin 9.2 9.0 0.2 TA 7.7 7.0 0.7 EGCG 8.1 7.0 1.1 Catechol 9.5 8.01.5 PG 9.3 7.0 2.3 HHQ 9.1 6.0 3.1

Based on the data from Examples 7-10, no one pH exists as an optimalcondition for forming polyphenol-based coatings. Additionally, based onthe data from Table 7, the optimal pH cannot be determined by relatingcoating pH to the pK_(a) of the precursor molecule. Taking these twoobservations into account, the overall theme is that each molecule mustbe optimized independently.

Example 11 Stability of Phenolic Coatings

In order to determine the stability of phenolic coatings in aqueousconditions, coated samples were incubated in buffers for several days.Tannic acid was dissolved at a concentration of 1 mg/mL in solutions of100 mM bis-Tris and 600 mM NaCl at pH 7. Three polycarbonate sampleswere submerged in the tannic acid solutions, rocked for 24 h, rinsedthoroughly with water, and dried with N₂ gas. The coating thickness wasdetermined via ellipsometry. Coated samples were then submerged in 50 mMbuffer solutions at pH 3 (formate), 5 (acetate), 7 (PBS), or 9 (bicine).At day 1, 4, and 7, samples were removed from buffers, rinsed thoroughlywith water, and dried with N₂ gas. The coating thickness that remainedwas calculated via ellipsometry, and stability was based on thepercentage of the initial coating thickness that was present at day 1,4, and 7 (FIG. 22).

Following a similar procedure, the stabilities of coatings based onpyrogallol, catechol, catechin (FIG. 23), HHQ, and EGCG wereinvestigated.

Example 12 Effect of Salt on Coating Formation

The effect of salt composition on coating formation was investigated atoptimal pH conditions identified previously for each molecule. As anexample, gallic acid was dissolved at a concentration of 1 mg/mL insolutions of 100 mM bis-Tris at pH 7 containing 600 mM NaCl, 600 mMMgCl₂, 600 mM CaCl₂, 100 μM CuCl₂ with 600 mM NaCl, or 100 μM ZnCl₂ with600 mM NaCl. Pieces of titanium dioxide (TiO₂) or silicon dioxide (SiO₂)were submerged in the gallic acid solutions, rocked for 48 h, rinsedthoroughly with water, and dried with N₂ gas. The coating ability wasassessed via x-ray photoelectron spectroscopy (XPS) by monitoring thedegree to which the substrate signal—Ti2p for TiO₂ and Si2p for SiO₂—wasdiminished (Tables 8-9). The results indicate that gallic acid solutionsproduced coatings on SiO₂ and TiO₂ in buffers containing MgCl₂ or CaCl₂,but not in NaCl-based buffers. None of coating precursor molecules wereable to coat SiO₂ when dissolved in NaCl-based buffers.

As before, the interpretation of the data is straightforward and basedupon photoelectron escape from the sample. As a coating is deposited ontop of the substrate, the photoelectrons representing the substrate areattenuated. Therefore the lower the substrate signal, the better themolecule was able to form a coating. Coatings greater than approximately10 nm in thickness entirely eliminate the signal from the substratebecause the photoelectrons cannot escape and be detected. Using thisstrategy, gallic acid, tannic acid, catechin, epigallocatechin gallate(EGCG), quercetin, phloroglucinol, pyrogallol (PG), catechol,resorcinol, hydroquinone, hydroxyhydroquinone (HHQ), and phenol—allmolecules tested—were able to coat both TiO₂ and SiO₂ with at least oneof the experimental conditions described here.

TABLE 8 Ti2p signal strength after immersing TiO₂ in buffered solutioncontaining precursor molecules. Ti2p Signal Strength (%) Molecule Na⁺Mg²⁺ Ca²⁺ Cu²⁺ Zn²⁺ Gallic acid, pH 7 107.6 0.0 0.0 106.3 80.7 Tannicacid, pH 6 e 0.0 0.0 e e Tannic acid, pH 7 0.0 88.9 11.4 0.0 0.0Catechin, pH 9 0.0 0.0 0.0 0.0 0.0 EGCG, pH 7 0.0 0.0 0.0 0.0 13.0Quercetin, pH 9 83.9 54.6 0.0 94.4 77.6 Phloroglucinol, pH 8 94.8 84.7106.8 94.7 91.9 Phloroglucinol, pH 9 88.6 51.6 81.7 94.4 111.9 PG, pH 712.6 0.0 0.0 0.0 3.9 Catechol, pH 8 9.7 0.0 0.0 89.8 93.5 Resorcinol, pH8 97.3 0.0 111.6 84.1 104.7 Hydroquinone, pH 9 80.8 62.5 0.0 93.5 108.7HHQ, pH 6 0.0 0.0 0.0 14.1 5.0 Phenol, pH 8 78.5 0.0 106.8 78.7 86.4^(a) Na⁺ = 600 mM NaCl; Mg²⁺ = 600 mM MgCl₂; Ca²⁺ = 600 mM CaCl₂; Cu²⁺ =100 μM CuCl₂ + 600 mM NaCl; Zn²⁺ = 100 μM ZnCl₂ + 600 mM NaCl. ^(b) pH 6& 7 = 100 mM bis-Tris; pH 8 & 9 = 100 mM bicine. ^(c) All coatingprecursors were dissolved at 1 mg/mL. ^(d) Values associated withsuccessful coatings (at least 20% signal reduction) are in bold. eRepresents conditions that were not investigated.

TABLE 9 Si2p signal strength after immersing SiO₂ in buffered solutioncontaining precursor molecules. Si2p Signal Strength (%) Molecule Na⁺Mg²⁺ Ca²⁺ Cu²⁺ Zn²⁺ Gallic acid, pH 7 95.3 0.0 7.7 91.0 107.5 Tannicacid, pH 6 e 83.8 102.7 e e Tannic acid, pH 7 111.9 50.3 105.4 103.090.6 Catechin, pH 9 107.1 0.0 99.2 105.7 81.7 EGCG, pH 7 108.5 20.6 99.8107.1 104.1 Quercetin, pH 9 90.2 7.8 0.0 88.5 95.1 Phloroglucinol, pH 885.3 69.4 91.7 83.9 88.7 Phloroglucinol, pH 9 30.5 76.5 84.6 78.9 90.4PG, pH 7 100.1 0.0 2.2 106.5 108.7 Catechol, pH 8 108.7 0.0 0.0 72.476.9 Resorcinol, pH 8 89.9 71.6 88.7 76.9 90.6 Hydroquinone, pH 9 85.977.1 86.5 68.2 89.0 HHQ, pH 6 94.9 80.5 1.5 93.5 104.8 Phenol, pH 8 93.00.0 70.8 71.9 85.6 ^(a) Na⁺ = 600 mM NaCl; Mg²⁺ = 600 mM MgCl₂; Ca²⁺ =600 mM CaCl₂; Cu²⁺ = 100 μM CuCl₂ + 600 mM NaCl; Zn²⁺ = 100 μM ZnCl₂ +600 mM NaCl. ^(b) pH 6 & 7 = 100 mM bis-Tris; pH 8 & 9 = 100 mM bicine.^(c) All coating precursors were dissolved at 1 mg/mL. ^(d) Valuesassociated with successful coatings (at least 20% signal reduction) arein bold. e Represents conditions that were not investigated.

In this Example, we included other salts, salt concentrations and pHranges employed during coating solution. FIG. 24 shows the effect ofsodium chloride (NaCl) concentration on PG-based coating thickness.Conditions: 2 mg/mL PG in 100 mM bis-Tris at pH 7 with varying amountsof NaCl.

FIG. 25 shows the effect of sodium chloride (NaCl) concentration and pHon PG-based coating thickness. Conditions: 2 mg/mL PG in 100 mM buffer(bis-Tris or bicine, depending on pH) with varying pH and varyingamounts of NaCl. The numbers at the top of the figure correlate coatingthickness to color.

FIG. 26 shows the effect of salt choice on PG-based coating thickness.Conditions: 2 mg/mL PG in 100 mM bis-Tris at pH 7 with 600 mM salt.Coating conditions: 2 mg/mL PG in 100 mM bis-Tris with salt at pH 7, 8h. All salts were at a concentration of 600 mM, except CuCl₂ and ZnCl₂.These two salts were added at a concentration of 100 μM to 600 mM NaCl.

FIG. 27 shows the effect of magnesium chloride (MgCl₂) concentration onPG-based coating thickness. Conditions: 2 mg/mL PG in 100 mM bis-Tris atpH 7 with varying amounts of MgCl₂.

Example 13 Successful Modification of Porous Membrane Substrates

Pyrogallol (PG) and tannic acid (TA) were used to modify poly(etherether ketone) (PEEK) membranes. PEEK samples were modified with 2 mg/mLPG or TA in 100 mM bicine and 600 mM NaCl at pH 7.8 for 24 h. Since PEEKand PG- and TA-based coatings are composed of carbon and oxygen, asubstrate-specific element was unavailable to confirm coatingdeposition. However, successful coating deposition was confirmed byX-ray photoelectron spectroscopy (XPS), observing the decrease in thecarbon-to-oxygen (C/O) ratio, as determined by C1s and O1s signals forcarbon and oxygen, respectively (Table 10). The experimental C/O ratioof samples decreased significantly following modification of PEEK withPG and TA. Compared to the theoretical value for the initial, baresubstrate (6.333), the C/O ratios after modification approach thetheoretical values for molecular PG or TA.

TABLE 10 Experimental (determined by XPS) and theoreticalcarbon-to-oxygen ratios (C/O) Experimental Theoretical Substrate C/OC/O* PEEK 6.001 6.333 PEEK + PG 2.526 2.000 PEEK + TA 1.894 1.652 *Thetheoretical C/O values are based on the molecular structure for PEEK,PG, and TA.

Macroscopic observations of water droplets on PEEK membranes indicatedenhanced wettability following modification with PG or TA (FIG. 28).Moreover, the wettability appears to be reversed after subsequentincorporation of silver nanoparticles reduced in-situ, as seen by thereduced spreading of water droplets on treated surfaces (FIG. 28).

Scanning electron microscopy (SEM) was employed to assess the surfacemorphologies of bare, PG- or TA-modified, and silvernanoparticle-functionalized PEEK membranes. The native porous structureof PEEK membranes is maintained following modification with PG and TA,both with and without further incorporation of silver nanoparticles(FIG. 29). The presence of silver nanoparticles on TA- and PG-modifiedPEEK membranes further confirms successful modification of themembranous material (FIG. 29).

Example 14 Comparison of Bacterial Viability on Coatings Based on TannicAcid and Dopamine

Unmodified polystyrene (as opposed to tissue culture plastic) wasmodified with coatings derived from tannic acid (TA) and dopamine (DA).TA was coated via a 2 mg/mL solution containing 100 mM bicine and 600 mMNaCl at pH 7.8. DA was coated via a 2 mg/mL solution containing 10 mMTris at pH 8.5. Polystyrene was coated with TA and DA for 24 h.Antimicrobial activity was tested against Candida albicans (fungus),Pseudomonas aeruginosa (Gram-negative bacteria), and Staphylococcusaureus (Gram-positive bacteria) (FIG. 30). Microbial specimens wereseeded at a density of 1.5×10⁶ colony forming units (CFU) per mL. After24 h, microbial viability was tested by staining with Syto 9 (for livecells) and propidium iodide (for dead cells). The fractions of live anddead cells were determined based on the area of fluorescence associatedwith the live and dead stains.

Example 15 Ability of (−)-Epicatechin to Form Coatings Based onConditions Used for (+)-Catechin

Catechin and epicatechin are stereoisomers. Until now, catechin has beeninvestigated for its ability to form coatings. This example demonstratesthat epicatechin is able to form coatings under conditions optimized forcatechin. Solutions of (+)-catechin (Ctn) and (−) epicatechin (ECtn)were made at a concentration of 1 mg/mL in 100 mM bicine and 600 mM NaClat pH 9. Polycarbonate (PC) samples were submerged in the solutions,rocked for 48 h, rinsed thoroughly with water, and dried with N₂ gas.The resulting coatings were stained with 100 mM solution of AgNO₃ for 48h, rinsed thoroughly with water, and dried with N₂ gas. Images were thencaptured with a digital camera (FIG. 31).

Example 16 Removal of Heavy Metal Ions from Solution by PhenolicCoatings

Pyrogallol (PG) and tannic acid (TA) were used to modify cotton gauzesamples weighing 140 mg±5%. Bare gauze and gauze modified with PG or TAwere incubated in 10 mL of 0.42 mM (˜72 μg/mL) AgNO₃ for 48 h, followingwhich the solutions were analyzed for silver content usinginductively-couple plasma mass spectrometry (ICP-MS). PG- andTA-modified gauze reduced silver concentration in solution, relative tounmodified gauze, by 53% and 83%, respectively (FIG. 32).

Example 17 Immobilization of Proteins and Enzymes onto Phenolic Coatings

Pyrogallol (PG) and tannic acid (TA) were used to modify TiO₂ samplesunder buffered saline conditions (pH 7.8, 100 mM bicine, 600 mM NaCl).Bare and phenolic-modified surfaces were exposed to 10 mg/mL lysozyme(from chicken egg white) solvated in water or buffered saline for 10minutes under mild agitation. Lysozyme-treated samples were thoroughlyrinsed with water and incubated in 0.1 M sodium dodecyl sulfate (SDS)for 30 minutes. The SDS incubation step was employed to screen outweakly-adhered enzyme from the surfaces. Lastly, the samples werethoroughly rinsed with water and dried under a nitrogen gas stream.

Bare and modified substrates were analyzed using X-ray photoelectronspectroscopy (XPS) for presence of protein. The N1s signal wasmonitored, the increase of which corresponded to deposition of a proteinon a surface (FIG. 33). TiO₂+PG surfaces yielded 95% and 51% increasesin immobilized protein content when compared to bare TiO₂, using waterand buffered saline for lysozyme deposition, respectively. TiO₂+TAsurfaces yielded 54% and 55% increases in immobilized protein contentwhen compared to bare TiO₂, using water and buffered saline for lysozymedeposition, respectively.

Example 18 Antioxidant Properties of Polyphenol-Derived Coatings

Pyrogallol (PG) and tannic acid (TA) were used to modify polystyrenesamples. Following modification, NIH 3T3 fibroblasts were cultured onbare and PG- or TA-treated surfaces. After 24 h of culture, the cellswere loaded with 2′,7′-dichlorofluorescin diacetate, followed bystimulation with a reactive oxygen species (ROS) inducing agent. Thefraction of ROS production was determined as a function of fluorescenceof the converted intracellular dichlorofluorescin (FIG. 34). It wasnoted that PG coatings were not able to substantially reduce ROSproduction relative to bare polystyrene controls. Tannic acid coatings,however, resulted in approximately 60% reduction in ROS productionversus bare polystyrene controls.

Example 19 Anti-Inflammatory Properties of Polyphenol-Derived Coatings

Pyrogallol (PG) and tannic acid (TA) were used to modify polycarbonate(PC) samples under buffered saline conditions (pH 7.8, 100 mM bicine,600 mM NaCl). RAW 264.7 murine monocytes were cultured on bare and PG-and TA-modified PC for 24 h, followed by stimulation with 1 μg/mL oflipopolysaccharides (LPS) derived from Pseudomonas aeruginosa for 1 h.The culture media was collected and analyzed for tumor necrosis factoralpha (TNF-α) using a commercially available ELISA kits (Invitrogen).TNF-α is a pro-inflammatory signaling molecule, elevated levels of whichare directly correlated to enhanced inflammatory response. Stimulationwith LPS results in upregulation of TNF-α production by RAW 264.7monocytes when compared to non-stimulated controls. Incorporation of aPG or TA coating mitigates the observed TNF-α production by at least60%, when compared to bare PC controls (FIG. 35).

In summary, we have described a facile, surface-independent, polyphenolcoating whereby substrates of all kinds contacted with the coatings ofthe present invention are modified to support at least one functionalreactive moiety on the substrate's surface. In some embodiments, thenitrogen-free phenolic compounds used in the present invention may beselected from epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC)and epicatechin gallate (ECG), tannic acid, gallic acid and pyrogallol.The reactive moiety reacts with and is bound to the coated surface. Thereactive moiety may comprise a metal ion selected from the groupconsisting of silver and gold ions, or a nucleophile selected from thegroup consisting of a protein or a thiol or amine containing polymer.Methods of use and kits comprising the coating are also included. Ingeneral, the method comprises contacting at least a portion of thesubstrate with the plant-derived or synthetic polyphenol coating of thepresent invention to provide a surface modified to support at least onereactive moiety.

The above description and attached figures are intended to beillustrative and not limiting of this invention. Many themes andvariations of this invention will be suggested to one skilled in thisand, in light of the disclosure. All such themes and variations arewithin the contemplation hereof. For instance, while this invention hasbeen described in conjunction with the various exemplary embodimentsoutlined above, various alternatives, modifications, variations,improvements, and/or substantial equivalents, whether known or that rareor may be presently unforeseen, may become apparent to those having atleast ordinary skill in the art. Various changes may be made withoutdeparting from the spirit and scope of the invention. Therefore, theinvention is intended to embrace all known or later-developedalternatives, modifications, variations, improvements, and/orsubstantial equivalents of these exemplary embodiments.

We claim:
 1. A method of forming a coating on a substrate surface, themethod comprising contacting at least a portion of the substrate surfacewith an aqueous solution comprising an effective amount of one or morenatural or synthetic nitrogen-free phenolic compounds, whereby a coatingforms on the substrate surface.
 2. The method of claim 1, wherein theone or more nitrogen-free phenolic compounds are selected from the groupconsisting of pyrogallol (PG), epigallocatechin-3-gallate (EGCG),epigallocatechin (EGC), epicatechin gallate (ECG), tannic acid (TA),hydroxyhydroquinone (HHQ), catechin, morin, quercetin, naringenin,naringin, rutin, phloroglucinol, catechol, resorcinol, hydroquinone,phenol, gallic acid, and isomers thereof.
 3. The method of claim 1, themethod further comprising one or more salts.
 4. The method of claim 3,wherein the one or more salts are selected from the group consisting ofa sodium salt, a potassium salt, a calcium salt, a magnesium salt, acopper salt, and a zinc salt.
 5. The method of claim 3, wherein the oneor more salts are selected from the group consisting of NaCl, NaNO₃,Na₂SO₄, KCl, K₂SO₄, MgCl₂, CaCl₂, CuCl₂, and ZnCl₂.
 6. The method ofclaim 1, wherein the aqueous solution comprises more than one salt. 7.The method of claim 1, wherein the aqueous solution further comprises abuffer.
 8. The method of claim 1, wherein the pH of the aqueous solutionis from 3.0 to 9.0.
 9. The method of claim 8, wherein the pH of theaqueous solution is from 6.0 to 9.0.
 10. The method of claim 1, themethod further comprising the step of contacting the resulting coatingwith a reactive moiety, whereby the reactive moiety reacts with andbecomes bound to the coating.
 11. The method of claim 10, wherein thereactive moiety comprises a nucleophile or a metal ion.
 12. The methodof claim 11, wherein the metal ion is selected from the group consistingof silver and gold ions.
 13. The method of claim 11, wherein thenucleophile is comprised of an amine- or thiol-functionalizedpoly(ethylene glycol) (PEG).
 14. A coating on a substrate surface asproduced by the method of claim
 1. 15. A method of providing antioxidantproperties to a substrate comprising depositing an antioxidant coatingon a substrate surface according to the method of claim 1, whereby thecoated substrate reduces the concentration of radicals and/or reactiveoxygen species.
 16. A method of providing anti-inflammatory propertiesto a substrate comprising depositing an anti-inflammatory coating on asubstrate surface according to the method of claim 1, whereby the coatedsubstrate exhibits a attenuated acute inflammatory response throughreduction of pro-inflammatory factors.
 17. A method of inhibitingmicrobial growth on a substrate comprising depositing a coating on asubstrate surface according to the method of claim 1, whereby the coatedsubstrate effectively kills microbes on contact or inhibits microbialgrowth.
 18. A method of removing metal ions from a liquid, comprisingcontacting the liquid with the coating of claim 15, whereby at leastsome of the metal ions in the sample are captured by the coating andremoved from the liquid sample.
 19. The method of claim 18, wherein themetal ions are selected from the group consisting of lead, cadmium,copper, mercury, and chromium.